A few days ago, I posted a message on the fact that a 54 residue peptide
containing 12 cysteine, alkylated with vynilpyridine, could not be digested
with trypsin (this is a peptide that my wife is studying)
I would like first to thank all the persons that did answer, and sorry if I don't
answer personally to each of you, but I thought a summary would be better.
Some of the advices were :
- test the stability by following unfolding with various amounts of urea
using CD or trp fluorescence.
My comment : what if the peptide only unfolds at 2M urea ? trypsin will not
be active anymore ...
- modifed cysteines immediately after lysines and argines : this is not
the case in this peptide
- test activity of trypsin : it was checked on parallel on a linear peptide
and it did work
- concentration of trypsin used : my wife used a 1:1 ratio of peptide:trypsin
at 37 degrees
- is the peptide a trypsin inhibitor ? no, see below
- aminomethylation of the cysteine instead of VP, which could then introduce
new cleavage sites : would you have references on that ?
My wife finally found conditions in which the peptide could be cut : she
added 0.5 % triton to the solution, and she got the 5 fragments she was
looking for. Hope this can be of some help to other people studying this
sort of very stable peptides
Thanks again for your help
Patrice Koehl, UPR 9003 du CNRS, Strasbourg, France
koehl at bali.u-strasbg.fr
